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APPLICATION OF THE SERIAL DILUTION TECHNIQUE
TO ESTIMATE THE BIOMASS OF NZ-FIXING BLUE-GREEN zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
ALGAE UNDER FIELD CONDITIONS
P.k REYNAUD
Office de la recherche scientifique et technique
Outre-Mer (ORSTOM), zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Dakar, Senegal
Abstract
The serial-dilution method developed to estimate algal biomass in
field samples is described. This method is illustrated by a transect
experiment in zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAa dry rice field.
It shows a log-normal distribution law
for algal material. The effect of taxon volume unit, enumeration and
-..
sampling on the accuracy of the method is determined. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
.
------ INTRODUCTION
The main problem in the measurement of the effect of inoculation
with blue-green algae in rice fields is to determi'ne the principal
factors which might be involved in the inoculation effect. The review
by Roger and Kulasooriya (1980) indicates that most work on algalization
has been performed to compare the grain yield in treatments inoculated
or non-inoculated with algae. Experiments conducted on this "black bQx"
basis give no additional information on the qualitative and quantitatiye
development of the algal inoculum and of the phototrophic nitrogen--
fixing activity although these parameters are important and may havg
explained why the algalization effect was positive, negative or
residual. To have a better understanding of the evolution of blue-green
algae during the rice cultivation cycle, it is necessary to estimate the
'
total algal biomass along the development of the crop from seedling
stage to harvest.
Algal abundance haa been estimated by three principal methods:
I
direct observation, measurement of pigments, and plating techniques.
The direct microscopic examination is generally used for qualitative
determinations, whereas the pigment analysis does not indicate the
composition of the algal flora. Platigg'techniques, which are more
frequently used, are advantageous in providing both quslitative and
.I
ORSTO~A Fonds DaCumeniaire
NO: zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAjil.3ns J;?rA 1$9
Gate : 6 \
-
quantitative information simultaneously, but the accuracy of such a
method depends on the dilution procedure.
The purpose of the present paper, is to describe an improved
serial-dilution method developed Ln pur lab, to estimate alggl biomass
in field samples.
1. HETHOD
The experiment was conducted in a dry rice field just after
cropping; 57 samples coinsisting of three soil cores of ons cm diameter
and about 1 cm depth each were collected alon,g a transect of 18.5 m.
They were stored in closed and dry bottles under lab temperature.
Each sample was zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAweighed, crushed and resuspended in 30 ml deionised
water arid thoroughly stirred. Total nitrogen, protein concentration,-
and pH of each suspension was measured. The suspension was dilutqd from
10-1 to zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAin glass tubes and one ml of each dilution was spread
onto a petri dish containing 30 ml of algal medium solidified with 1%
agar. We used the BG 11 medium (Allen and Stan,iar 1968) for the
IO-’ and dilutions tp enumerate the total algal constituants
and the BG 11 medium minus nitrogen with low5
dilutions to enumerate specifically nitrogen fixing blue-green algae.
For each dilution three petri dishes Were used. Incubations were
lconducted in a light chamber (500 lux, 30°C) for 21 days. Counts
were performed with a stereomicroscope WILD YS. The petri dish wag
divided by a frame into 1 cm squares, and each square was examined under
a x 12 magnification. Enumeration of algsl colonies Was perfOrW34 on
the entire surface of each petri dish.
II; RESULTS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
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Ji- vO32!!!!LUP@i
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In this method of enumeration each algal colony is presumed to
have arisen from a propagule which could either be a short piece of
filament or an akinete. To conrert the algal numbers to biomass, it
is necessary to calculate the mean volume of this propagule unit and
J
the following method was adopted for this purpose. From these
enumerations, 14 principal taxons were identified (Table 11, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
12Q t zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
I
I
Several colonies belonging to each taxon were picked frop the petri
dish and vigourously stirred for 10 minutes. With this suspension
we measured, under microscope, the size of a hundred pieces of
l
filament-or the diameter of cell aggregates. From these
measurements, a volume unit was calculated for each taxon by a
assuming that the form of the broken filaments corresponded roughly
to either a cylinder, sphere or cone. The value of each taTon
volume unit is thus specific to the algae collected in this i
ecosystem but can be used for other ecosystems if the taxon is well .I ,
identified,
The relative error determined here for r=5% is closely related I
to the ability to fragment the colonies and to the dispersion of
broken filaments during the stirring and diluting steps. For
instance the size of filgments of Nostoc Punctiforme (relatiye error zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
= 9%) is more homogenous than those of filaments of Anabaena
- skherica (relative error = 20%) and the relative error of the
estimation of this taxoq biomass would be higher than for N.
psnc t i f o qm. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAzyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
I 'L .
B; Enumerat/o,n of taxons zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
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Enumeration of taxons was normally conducted using two succesive
dilutions and the resul$ was expressed as the mean of the six petri
(tishes
I .1
On the medium BG 11 minus nitrogen, eucaryotic algae and non
nitrogen fixing blue-green algae grew normally during the first
week, then their growth stopped and colonies became yellow
confirming their inability tq fix N2 (Reynaud and Roger $9771,
>.
For N2-fixing blue-green algae (Np-fix BGA) there was a good
correlation between the number of colonies which developed, either
qn mediuq with or without nitrogen source.
!'
Tho, use of tbe two media and three dilutions allowed us tg: zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
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- distinguish slow growing taxons 'fpom fast growing ones I
- count each taxon as ita optimal dilution I
- reduce.possible zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAcompetition between algal groups
- easily isolate each taxon,
Petersen (1932) pointed out that the dilution method wag not
reliable for filamentous type and that some spreading types also do
not form individual colonies in the agar medium. To have an
estimation of these biomasses we have settled on a scale of the
denLity of separation of filaments for these taxons on 1 cm2 of
agar plate; we have compared it with a direct enumeration of
Means zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
filaments under microscope for a auspension of Lyngbya sp.
triplicates estimations were identical with the two methods.
C. @3_qmasu determination
The accuracy of algal estimation and in situ ARA measurements
depend upon the density of sampling and of the distribution law of
the variable. Earlier studies on the correlation between means and
variances of enumeration of soil microorganisms and in situ ABA
measurements, indicates that these variables have approximately a
log-normal distribution (Roger & &., 1977,,Roger and Reynaud,
1978). The firsit implication of this law was that the confidence
interval and parametric statistical variable (i.e.: t variable of
Student Fisher) must be calculated using the logarithms of algal
enumerations or AR4 measurements. The confidence interval W,ES zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
dissymmetrical, its inferior limit was generally slightly lower than
that incorrectly calculated using the t variable of Student Fisher;
The validity of the zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
the upper limit was generally higher.
logarithmic transformation must be checked by a method bssed on the
ratio between two correction coefficients established by Ney" and
Scott (1960) and programmed by Roger et al. (1978); transformation
is considered 8s valid when the ratio c-/c is include4 between
0.66 and 1.33.
I
The transect experiment is a good demonstration of the log--
normal distribution law for algal material. When variables such as
pH, weight of samples and N concentration in soils, are distributed
along the transect as a normal distribution, the log-normal
transformation is justifiable fop'l0 taxons out of 11 (Table 2). In
fact, &abg= sphaerica has a c-/c ratio of 3.465 depending of
,
122
1 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
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